Volume 143, Issue 1, Pages 155-165.e8 (July 2012) An RNA Aptamer That Binds Carcinoembryonic Antigen Inhibits Hepatic Metastasis of Colon Cancer Cells in Mice  Young Ju Lee, Seung Ryul Han, Nam Yeon Kim, Soo–Han Lee, Jin–Sook Jeong, Seong–Wook Lee  Gastroenterology  Volume 143, Issue 1, Pages 155-165.e8 (July 2012) DOI: 10.1053/j.gastro.2012.03.039 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Sequences and structural determinations of selected RNA aptamers that bind to the CEA. (A) Sequence of selected RNAs. Selected RNAs can be divided into several different groups of very similar RNA sequences and some of them were present multiple times (numbers in parentheses). The lines drawn represent that nucleotides found at these positions were identical. Selected sequences are denoted by italics. (B) Enzymatic mapping of the secondary structure of the selected RNA group I. RNA was enzymatically digested with RNases T1 (lane 2), nucleases S1 (lane 3), and RNases V1 (lane 4). The partially digested products were then separated on a 12% polyacrylamide gel with urea along with a partial alkaline hydrolysis ladder (lane 1, AH). (C) Enzymatic footprinting of CEA-group I RNA complex. The RNA was incubated in the absence (lanes 2, 6, 11) or presence of increasing amounts of CEA (162 fmole in lanes 3, 7, 12; 486 fmole in lanes 4, 8, 13; 1,458 fmole in lanes 5, 9, 14). Protein−RNA mixtures were digested with RNases T1 (lanes 2−5), nucleases S1 (lanes 6−9), and RNases V1 (lanes 11−14). The sites protected from each nuclease by the protein are indicated by bars on the right side. (D) The computer-predicted model of the secondary structure of group I RNA. Digestion patterns of the RNA to various nucleases are mapped. Squares, triangles, and circles indicate RNases T1, S1 nucleases, and RNases V1 cleavage sites, respectively. Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Minimization of group I RNA and specific binding to metastasis-inducing domain of CEA. (A) Minimized sequence of group I RNA aptamer and loop mutant aptamer. (B) The computer-predicted model of the secondary structure of minimized group I RNA series. (C) An electrophoretic mobility shift assay was used to examine protein-YJ-1 aptamer interaction. YJ-1 aptamer was incubated in the absence (lane 1) or presence of NCEA (lane 2 and 3) or ACEA (lane 4), or N domain (lane 5), A1 domain (lane 6), N+A1 domain (PELPK, lane 7), or mutant N+A1 domain (RELSK, lane 8) of CEACAM5. (D) Inhibition of CEA uptake by YJ-1 aptamer. P388D1 cells were transfected with heterogeneous nuclear ribonucleoprotein M4, and then treated with fluorescein-labeled CEA with YJ-1 or mutant aptamer. The value of CEA uptake was assessed with 3 independent experiments. Uptake level of fluorescein-labeled CEA in YJ-1 aptamer- or mutant aptamer-treated cells was compared with untreated control cells. ***P < .0001, **P < .005. Error bars, ±standard deviation. Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Inhibitory effect of YJ-1 aptamer on cell migration. Cell migration degree of CEA positive cell lines, LS174T (A) and LoVo (B), or CEA-negative cell line, HT-29 (C), was measured in the absence or presence of YJ-1 or a mutant aptamer. Migration of untreated cells was used as a positive control and set as 100%. Values represent means of 6 independent experiments. ***P < .0001. Error bars, ±standard deviation. Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Inhibitory effect of YJ-1 aptamer on homotypic cell aggregation. Cell aggregation was measured in CEA-positive cell lines, LS174T (A), CAPAN-1 (B), and LoVo (C), or CEA negative cell lines, HT-29 (D), and MCF7 (E), treated with YJ-1 or mutant aptamer in the presence or absence of Ca2+. Aggregation of untreated cells was used as a positive control. The average of 10 fields was recorded in 3 replicate studies for each cell line. **P < .005; ***P < .0001. Error bars, ±standard deviation. Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Specific inhibition of cancer cell invasion by YJ-1 aptamer. Cancer cell invasion through a polycarbonate membrane coated with collagen proteins was analyzed with CEA-positive cell lines, LS174T (A), CAPAN-1 (B), and LoVo (C), or CEA-negative cell lines, HT-29 (D) and MCF7 (E). Invasion of cells treated with YJ-1 or mutant aptamer was analyzed and represented as percentage of untreated control cells. FBS in the lower chamber served as the chemoattractant for invasion. Reduced invasion was noted in negative control samples that did not have FBS (without serum). Values represent means of 3 independent experiments. *P < .05; **P < .005; ***P < .0001. Error bars, ±standard deviation. Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Decrease in CEA-related anoikis resistance by RNA aptamer through direct blocking of CEA and DR5 interaction. (A) CEA-dependent anoikis resistance was analyzed by a caspase 8 activity assay. In suspension culture, the LS174T cell line was treated with YJ-1 or mutant aptamer, or etoposide (10 μg/mL). Caspase activity in each treated cell was represented as a percentage of untreated cells. (B) Effect of YJ-1 aptamer on in vitro binding of CEA to DR5. Purified DR5 and his-tagged CEA were incubated with YJ-1 or mutant aptamer and precipitated using Ni-NTA agarose bead. Bound protein was subjected to immunoblot analysis. Negative controls (without CEA) showed absence of nonspecific binding of DR5 protein to the Ni-NTA agarose bead. Input represents 5% of reaction mixtures. (C) CEA and DR5 interaction was inhibited by YJ-1 aptamer in cells. HEK 293T cells stably expressing YJ-1 or mutant aptamer were cotransfected with vectors encoding CEA and DR5 with the SEAP reporter plasmid. Protein−protein interactions were then assessed by measuring SEAP activity and represented values as relative to a control cotransfected with basal vectors and reporter construct (Basal). Values represent means of 3 independent experiments. *P < .05; **P < .005. Error bars, ± ±standard deviation. (D) Effect of YJ-1 on intracellular interaction of CEA and DR5. Cells stably expressing YJ-1 or mutant aptamer were cotransfected with vectors encoding CEA and FLAG-tagged DR5. Cell extracts were immunoprecipitated using anti-FLAG M2 affinity gel (IP: FLAG), and then subjected to immunoblot analysis. Each cell extract (3%) before the immunoprecipitation was used as an input control. Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Inhibition of CEA-mediated liver metastasis of colon cancer by RNA aptamer in animal model for metastasis. (A) A representative metastasized liver and spleen in a mouse after splenic injection of LS174T cells treated with YJ-1 aptamer or mutant aptamer. Mice injected with untreated cells (phosphate-buffered saline) were used as controls. (B) Tumor masses of YJ-1 or mutant aptamer-treated group and untreated group (n = 7 each) were determined and plotted. Mean tumor mass is presented with standard deviation. *P < .02. (C) Serum aspartate aminotransferase/alanine aminotransferase levels in YJ-1, mutant aptamer, and untreated groups were determined by enzyme activity assay (n = 7 each). Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 7 Gastroenterology 2012 143, 155-165.e8DOI: (10.1053/j.gastro.2012.03.039) Copyright © 2012 AGA Institute Terms and Conditions